Multiorgan neutrophilic inflammation in a Border Collie with “trapped” neutrophil syndrome

Abstract Trapped neutrophil syndrome is a rare congenital disease recognized in Border Collies and is characterized by persistent neutropenia with myeloid hyperplasia. The mechanism of neutropenia has not been described. We document the case of a young Border Collie diagnosed with trapped neutrophil syndrome based on clinical features, blood and bone marrow evaluation, and presence of the associated homozygous mutation. Results from flow cytometric and storage studies suggested lower neutrophil survival time. The dog had substantial neutrophilic inflammation in multiple organs, indicating that neutrophils could leave the marrow and enter tissues, making the term “trapped” neutrophil syndrome a misnomer.

ease has been characterized by persistent neutropenia with concurrent myeloid hyperplasia. 1,3,4,7 Persistent neutropenia in these dogs results in an impaired innate immune response with development of persistent or recurrent infections that involve the gastrointestinal tract, multiple joints, or a combination of both. [1][2][3][4][5] The disorder also manifests as musculoskeletal abnormalities including lameness, stunted growth, and an elongated, narrow muzzle. 4,5 The genetic mutation in TNS is the same as that characterized for Cohen syndrome in people 8 ; both disorders are caused by a mutation of the gene for vacuolar protein sorting-associated protein 13B (VPS13B). 8,9 The protein VPS13B localizes to the outer membrane of the Golgi and is essential for maintenance of the structural and functional integrity of the Golgi. 10 Cohen syndrome and TNS display similarities in clinical manifestations including slender extremities, craniofacial malformations, and neutropenia.
In this case report, we describe clinical, laboratory, and necropsy findings in a Border Collie diagnosed with TNS, provide evidence for shorter neutrophil survival in this dog, and document that many neutrophils can leave bone marrow and reach inflamed tissues, thus challenging the suitability of the name "trapped" neutrophil syndrome.

| CASE SUMMARY
A 3-month-old intact male Border Collie was presented to Michigan State University Veterinary Medical Center (MSU-VMC) for a 2-day history of pyrexia, lethargy, and lameness progressing to nonambulation. The dog was reported to have episodic signs of gastrointestinal disease and "failure-to-thrive" since 6 weeks-of-age. Three weeks before presentation, the dog was hospitalized at the referring veterinarian with vomiting, diarrhea, hematochezia, and dehydration. The dog was vaccinated with a SQ core vaccine (Vanguard DAPP/L4, Zoetis, Parsippany, New Jersey) and an intranasal bordatella vaccine (Vangard B intranasal, Zoetis, Parsippany, New Jersey) 18 days before presentation. A cage-side parvovirus antigen test (Supplemental Information) and a fecal flotation test were negative. Initial CBC and serum chemistry results reportedly revealed nonregenerative anemia and neutropenia with no other relevant findings. The dog received inpatient supportive care for gastroenteritis and was discharged the next day with instructions to administer amoxicillin.
Three weeks later, the dog was presented to the MSU-VMC because of fever, lethargy, and an inability to rise. On physical examination, the dog had dull mentation and was pyrexic (106.2 F) and tachycardic (160 bpm). The mandibular lymph nodes were enlarged and firm. Multiple joints were enlarged and warm, and the dog was nonambulatory. The dog had an abnormally narrow and elongated muzzle and abnormally slender extremities ( Figure 1) Abdominal ultrasonography revealed an enlarged hyperechoic liver, an enlarged heterogeneous spleen, and abdominal lymphadenomegaly; the right colic lymph node contained anechoic material. Aspirates of the liver, spleen, abdominal lymph nodes, mandibular lymph node, and synovial fluid were evaluated cytologically. Hepatic Given the signalment, elongated narrow muzzle and slender extremities, history of recurrent illness, and persistent low-normal neutrophil concentration, the clinical suspicion for TNS was high and a blood sample was submitted for genetic testing on day 4 (Supplemental Information). To obtain quicker support for TNS and assess for other disease, bone marrow core and aspirate samples were collected from the right ileal wing on day 5. These samples collectively revealed marrow with 95% to 100% hematopoietic cellularity, a markedly increased myeloid to erythroid ratio (24 : 1), and therefore marked myeloid hyperplasia ( Figure 3). Maturation of neutrophil precursors was complete through  Based on the preceding results, a blood storage experiment was done to assess in vitro cell preservation over time. After 12-hour refrigeration of EDTA-anticoagulated blood, most neutrophils in a stained blood smear from the TNS dog had moderate-marked deterioration and were lysed or partially lysed with swollen nuclei ( Figure 6A). Monocytes were only mildly deteriorated, and lymphocytes were mostly well preserved ( Figure 6B,C). Compared to a 12-hour sample, lysed cells increased from 5% (350/μL) to 22% (1450/μL) at 36 hours; neutrophil deterioration had increased, but preservation of monocytes and lymphocytes was relatively F I G U R E 4 Histologic specimens of ileum (A), renal cortex (B), spleen (C), and liver (D and E). Effacing transmural necrotizing and suppurative lesion within the ileum, with the serosa to the left and the ulcerated mucosa to the right (A). Marked suppurative inflammation within the renal cortex and associated tubular degeneration (B). Marked amyloid deposition throughout this region of the spleen (C). Moderate hepatic amyloid deposition (asterisks denote representative accumulations) and many sinusoidal leukocytes, mostly neutrophils, despite a low-normal blood neutrophil concentration (D and E).  Expression of cell surface selectin, integrins, and CXCR4 did not appear abnormal in the TNS dog. Others have found no CXCR4 mutations in dogs with TNS. 14 Decreased neutrophil expression of CD62L and CD11b was shown for 1 person with Cohen syndrome, and it was attributed to increased neutrophil activation in vivo. 15 A limitation of our flow cytometric study, including annexin V, is that the affected dog was receiving prednisone (0.88 mg/kg/d), while controls were not. In cattle and people, glucocorticoids can reduce neutrophil expression of CD62L, 16,17 and in cattle, glucocorticoids can decrease neutrophil apoptosis, rather than increase it 18 ; the effects of glucocorticoid use on expression of canine neutrophil surface proteins is unclear.

ACKNOWLEDGMENT
Funding provided by BISSELL Pet Foundation. Acknowledgment to the Michigan State University Investigative HistoPathology Lab (Amy S. Porter, Amy E. Allen, Jessielee V. Neumann).